Sperm proteome and sperm parameters of captive Amazon squirrel monkeys (Saimiri collinsi Osgood, 1916) during nonfatted and fatted condition

Description

The present study evaluates the sperm proteome after the dilution of the seminal coagulum and the sperm parameters during nonfatted and fatted conditions in Saimiri collinsi (Amazon squirrel monkey). Thirty-five semen samples were collected by electroejaculation, and extracted proteins were trypsin-digested, desalted, and analyzed by LC-MS/MS. The Mascot software searches were performed on an in-house server against an online Saimiri boliviensis boliviensis database. Label-free LC-MS/MS allowed the identification of 1,161 proteins in sperm samples. Out of the 65 proteins that were differently expressed. The most important biological processes related to the upregulation of in animals of nonfatted condition were cellular processes and metabolic processes. The main molecular functions of these proteins were determined to be catalytic activity and binding. Sperm proteins were mainly defined as cellular anatomical entities and protein complexes, as cellular components. The protein classes identified in the study included metabolite interconversion enzyme, transport, chaperone, and protein modifying enzyme. In fatted condition, the most important biological processes related to the upregulation proteins were cellular processes, metabolic processes, and localization. The main molecular functions of these proteins were determined to be binding, catalytic activity, ATP-dependent activity, and transport activity. Regarding cellular components, the proteins were mainly defined as cellular anatomical entities and protein complexes. The study identified protein classes included cytoskeletal protein, metabolite interconversion enzyme, transporter, RNA metabolism protein, chaperone and Protein modifying enzyme. After analyzing the data using DAVID, we discovered that 20 genes were in nonfatted and 25 genes in fatted conditions. In nonfatted conditions associated with biological processes, molecular functions such as chaperone and cellular components such as cilium, flagellum, and cell projection. In fatted conditions, proteins were highly represented in biological processes such as fertilization, spermatogenesis, and transport, also involved in molecular functions such as chaperone and motor protein, and cellular components such as dynein, cytoskeleton, proteasome, cell projection, and cytoplasm. This represents the most comprehensive overview of the sperm proteome in the Amazon squirrel monkey sperm and will contribute to the elucidation of the underlying mechanisms associated with sperm function.

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Dried sperm sample was resuspended in 50 µL of lysis buffer (0.1 M Tris-HCl (pH 8.0), 4% sodium dodecyl sulfate, and 10 mM dithiothreitol; Sigma-Aldrich®, St. Louis, MO, USA) and centrifuged at 5000 g for 1 h at 4°C (NanoSpin Plus, Loccus biotecnologia, Cotia, São Paulo, Brazil). The supernatant was reserved for the preparation of suspension samples for bottom-up proteomic analysis with tryptic digestion, using the method established by Zougman et al. (2014). The extracted peptides were analyzed on an UltiMate 3000 RSLCnano/Q-Exactive system (Thermo Fisher Scientific, Bremen, HB, Germany) that was set up with a Nanospray Flex ion source. The tryptic peptides (~1 µg loaded) were separated on a 50 cm × 75 µm (i.d.) column (Thermo Fisher Scientific, Bremen, HB, Germany) using a 120 min gradient of 12–45% acetonitrile. The mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data were recorded using a standard data-dependent acquisition method, with the following conditions: m/z range of 300–1600; Automatic Gain Control targets of 3 × 106 (MS) and 5 × 104 (MS/MS); resolutions of 70 K (MS) and 35 K (MS/MS); dynamic exclusion set to 20 s, and normalized collision energy set to 28. Xcalibur software (v. 3.1; Thermo Fisher Scientific, Bremen, HB, Germany) was used to evaluate the raw data, which were converted to mgf format (for Mascot database searching) using the MS convert module of ProteoWizard (v. 3.0.9016, Palo Alto, CA, USA). The Mascot (Mascot software v. 2.6, Matrix Science Inc., Boston, MA, USA) searches were performed on an in-house server against an online Saimiri boliviensis boliviensis (Bolivian squirrel monkey) database (National Center for Biotechnology Information, Bethesda, MD, USA). MaxQuant software (v.1.6.1.0, Max-Planck-Institute of Biochemistry, Munich, BY, Germany) was used for the label-free quantification.

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